1. Field of the Invention
The present invention relates to a biosensor that can easily quantify a specific component in a sample liquid with speed and accuracy, and a method for measuring a concentration of a substrate (a specific component) in a sample by using the biosensor. More particularly, it relates to a biosensor that can quantify a specific component in a sample liquid by reacting the specific component in the sample liquid to an oxidoreductase that specifically reacts to the component and then by quantifying the change of concentration of a material that has changed through the reaction after a predetermined period of time, and to a method for measuring the concentration of a substrate in a sample by using the biosensor.
2. Description of the Prior Art
Various types of biosensors utilizing specific catalyses of enzyme have been recently developed. A saccharide biosensor will be described as an example of such biosensors as follows:
The optical rotation method, the colorimetric method, the reductimetry method and other methods using different kinds of chromatographies have been developed as methods for quantitative analysis of saccharides. However, none of these methods can provide high accuracy due to the relatively low specificity against saccharides. Additionally, the optical rotation method is easy to operate but is largely influenced by the operating temperature. Therefore, it is not appropriate for common use at home and the like.
The saccharides contained in fruit are generally assessed as saccharine degrees. A refractometer of the light refraction system is often used for quantifying the saccharine degree. This refractometer functions by utilizing change of the light refractive index caused by liquid concentration. Therefore, the refractometer of the light refraction system is influenced by all the components dissolved in the sample liquid, for example, by organic acid such as citric acid or maleic acid that is contained in fruit juice in large amounts when a saccharide in the fruit is quantified. Thus, accurate quantification by this refractometer is impossible.
A glucose sensor will now be described as an example of a biosensor used in a clinical field.
A conventional method for quantifying glucose contained in blood is to centrifuge blood taken from a patient and then to measure the thus obtained blood plasma. This method requires a lot of time as well as labor. Therefore, a sensor that can directly measure glucose in blood obtained from a patient is desired.
A sensor similar to a test paper for urinalysis has been developed as a simple glucose sensor. This glucose sensor comprises a support in a stick shape and a holder fixed to the support. The holder includes an enzyme reacting only to glucose and a dye, the color of which is changed by reacting with a production of the enzyme reaction. Blood is dropped onto the support of the glucose sensor and the change of the color of the dye after a predetermined period of time of the dropping is visually observed or optically measured, whereby the content of glucose in the blood can be measured. However, the quantifying method using this glucose sensor has low accuracy due to interference by the colored materials in the blood.
Japanese Laid-Open Patent Publication No. 1-291153 discloses the following glucose sensor with high accuracy as a method for quantifying a specific component in a sample liquid from a living body such as blood without diluting or stirring the sample liquid:
The biosensor comprises a base 42, and a spacer 3 and a cover 4 that are laminated integrally onto the base 42 as is shown in FIGS. 13 and 14.
The base 42 comprises an electrical insulating substrate 1, an electrode system 43 formed on the substrate 1 by screen printing, etc. and a reaction layer 44 provided on the electrode system 43. The electrode system 43 includes a working electrode 45 and a counter electrode 46 that are electrically insulated from each other by an insulating layer 47. The working electrode 45 and the counter electrode 46 are connected to leads 12 and 13 formed on the substrate 1, respectively.
The reaction layer 44 includes a hydrophilic polymer, an oxidoreductase and electron acceptors and covers the working electrode 45 and the counter electrode 46.
As is shown in FIG. 13, the spacer 3 is in a U-shape and has a groove 17. When the spacer 3 and the cover 4 are laminated on the base 42, a passage 18 through which a sample liquid passes is formed between the base 42 and the cover 4 as is shown in FIG. 14. One end of the passage 18 is open at one end of the base 42 and the opening serves as a sample supply port 23. The other end of the passage 18 is open on the cover 4 and the opening serves as an air port 24.
The operation of the glucose sensor with the above-mentioned structure is as follows: A sample liquid supplied through the sample supply port 23 reaches the reaction layer 44 through the passage 18 and the oxidoreductase and the electron acceptors contained in the reaction layer 44 are dissolved in the sample liquid. Thus, while an enzyme reaction is proceeded between a substrate in the sample liquid and the oxidoreductase, the electron acceptors are reduced. After finishing the enzyme reaction, the reduced electron acceptors are electrochemically oxidized. A value of an oxidation current obtained at this point provides a concentration of the substrate in the sample liquid.
However, the conventional biosensor has the following disadvantages:
The sample liquid may include reductive materials that can reduce the electron acceptors other than the substrate to be measured. Moreover, viscosity, etc. of the sample liquid to be measured vary.
Accordingly, in measuring a concentration of the substrate in the sample liquid including the substrate and another reductive material that can reduce the electron acceptors, the response values of the sensor are inconstant, and therefore, the reductive material should be eliminated before the measurement. Such a pretreatment results in increasing the number of steps in measuring the concentration of a substrate in a sample liquid.
Moreover, the sensor response also depends upon a measuring time. For example, an accurate concentration can not be obtained when the oxidation current is measured before completely finishing the reaction.
Furthermore, time required for the sample liquid to reach the reaction layer and the rate of the reaction of the substrate to the enzyme depends upon the viscosity of the sample liquid. Therefore, the inconstant viscosities result in inconstant sensor responses.